This invention relates to chemically synthesized ribozymes, or enzymatic nucleic acid molecules and derivatives thereof.
The following is a brief description of nucleic acid catalysts. This summary is not meant to be complete but is provided only for understanding of the invention that follows. This summary is not an admission that all of the work described below is prior art to the claimed invention.
Nucleic acid catalysts are nucleic acid molecules capable of catalyzing one or more of a variety of reactions including the ability to repeatedly cleave other separate nucleic acid molecules in a nucleotide base sequence-specific manner. Such enzymatic nucleic acid molecules can used, for example, to target virtually any RNA transcript (Zaug et al., 324, Nature 429 1986; Cech, 260 JAMA 3030, 1988; and Jefferies et al., 17 Nucleic Acids Research 1371, 1989).
Because of their sequence-specificity, trans-cleaving enzymatic nucleic acid molecules show promise as therapeutic agents for human disease (Usman and McSwiggen, 1995 Ann. Rep. Med. Chem. 30, 285-294; Christoffersen and Marr, 1995 J. Med. Chem. 38, 2023-2037). Enzymatic nucleic acid molecules can be designed to cleave specific RNA targets within the background of cellular RNA. Such a cleavage event renders the mRNA non-functional and abrogates protein expression from that RNA. In this manner, synthesis of a protein associated with a disease state can be selectively inhibited.
There are seven basic varieties of naturally-occurring enzymatic RNAs are known presently. Each can catalyze the hydrolysis of RNA phosphodiester bonds in trans (and thus can cleave other RNA molecules) under physiological conditions. Table I summarizes some of the characteristics of these ribozymes. In general, enzymatic nucleic acids act by first binding to a target RNA. Such binding occurs through the target binding portion of a enzymatic nucleic acid which is held in close proximity to an enzymatic portion of the molecule that acts to cleave the target RNA. Thus, the enzymatic nucleic acid first recognizes and then binds a target RNA through complementary base-pairing, and once bound to the correct site, acts enzymatically to cut the target RNA. Strategic cleavage of such a target RNA will destroy its ability to direct synthesis of an encoded protein. After an enzymatic nucleic acid has bound and cleaved its RNA target, it is released from that RNA to search for another target and can repeatedly bind and cleave new targets.
In addition, several in vitro selection (evolution) strategies (Orgel, 1979, Proc. R. Soc. London, B 205, 435) have been used to evolve new nucleic acid catalysts capable of catalyzing a variety of reactions, such as cleavage and ligation of phosphodiester linkages and amide linkages, (Joyce, 1989, Gene, 82, 83-87; Beaudry et al., 1992, Science 257, 635-641; Joyce, 1992, Scientific American 267, 90-97; Breaker et al., 1994, TIBTECH 12, 268; Bartel et al., 1993, Science 261:1411-1418; Szostak, 1993, TIBS 17, 89-93; Kumar et al., 1995, FASEB J., 9, 1183; Breaker, 1996, Curr. Op. Biotech., 7, 442).
The enzymatic nature of a ribozyme is advantageous over other technologies, since the effective concentration of ribozyme necessary to effect a therapeutic treatment is generally lower than that of an antisense oligonucleotide. This advantage reflects the ability of the ribozyme to act enzymatically. Thus, a single ribozyme molecule is able to cleave many molecules of target RNA. In addition, the ribozyme is a highly specific inhibitor, with the specificity of inhibition depending not only on the base-pairing mechanism of binding, but also on the mechanism by which the molecule inhibits the expression of the RNA to which it binds. That is, the inhibition is caused by cleavage of the RNA target and so specificity is defined as the ratio of the rate of cleavage of the targeted RNA over the rate of cleavage of non-targeted RNA. This cleavage mechanism is dependent upon factors additional to those involved in base-pairing. Thus, it is thought that the specificity of action of a ribozyme is greater than that of antisense oligonucleotide binding the same RNA site.
The development of ribozymes that are optimal for catalytic activity would contribute significantly to any strategy that employs RNA-cleaving ribozymes for the purpose of regulating gene expression. The hammerhead ribozyme functions with a catalytic rate (kcat) of xcx9c1 minxe2x88x921 in the presence of saturating (10 mM) concentrations of Mg2+ cofactor. However, the rate for this ribozyme in Mg2+ concentrations that are closer to those found inside cells (0.5-2 mM) can be 10- to 100-fold slower. In contrast, the RNase P holoenzyme can catalyze pre-tRNA cleavage with a kcat of xcx9c30 minxe2x88x921 under optimal assay conditions. An artificial xe2x80x98RNA ligasexe2x80x99 ribozyme has been shown to catalyze the corresponding self-modification reaction with a rate of xcx9c100 minxe2x88x921. In addition, it is known that certain modified hammerhead ribozymes that have substrate binding arms made of DNA catalyze RNA cleavage with multiple turn-over rates that approach 100 minxe2x88x921. Finally, replacement of a specific residue within the catalytic core of the hammerhead with certain nucleotide analogs gives modified ribozymes that show as much as a 10-fold improvement in catalytic rate. These findings demonstrate that ribozymes can promote chemical transformations with catalytic rates that are significantly greater than those displayed in vitro by most natural self-cleaving ribozymes. It is then possible that the structures of certain self-cleaving ribozymes may not be optimized to give maximal catalytic activity, or that entirely new RNA motifs could be made that display significantly faster rates for RNA phosphoester cleavage.
Chemically-modified ribozymes can be synthesized which are stable in human serum for up to 260 hours (Beigelman et al, 1995, J. Biol. Chem., 270, 25702) and maintain near wild type (chemically unmodified equivalent of modified ribozyme) activity in vitro. A number of laboratories have reported that the enhanced cellular efficacy of phosphorothioate-substituted antisense molecules. The enhanced efficacy appears to result from either i) increased resistance to 5xe2x80x2-exonuclease digestion (De Clercq et al., 1970 Virology 42, 421-428; Shaw et al., 1991 Nucleic Acids Res. 19, 747-750), ii) intracellular localization to the nucleus (Marti et al., 1992 Antisense Res. Dev. 2, 27-39), or iii) sequence-dependent non-specific effects (Gao et al., 1992 Molec. Pharmac. 41, 223-229; Bock et al., 1992 Nature 355, 564-566; and Azad, et al., 1993 Antimicrob. Agents Chemother. 37, 1945-1954) which are not manifested in non-thioated molecules. Many effects of thioated compounds are probably due to their inherent tendency to associate non-specifically with cellular proteins such as the Sp1 transcription factor (Perez et al., 1994 Proc. Natl Acad Sci. U.S.A. 91, 5957-5961). Chemical modification of enzymatic nucleic acids that provide resistance to cellular nuclease digestion without reducing the catalytic activity or cellular efficacy will be important for in vitro and in vivo applications of ribozymes.
The references cited above are distinct from the presently claimed invention since they do not disclose and/or contemplate the enzymatic nucleic acid molecules of the instant invention.
This invention relates to nucleic acid catalysts with one or more L-nucleotide-substitutions. These substitutions alone or in combination with other D- and L-chemical substitutions protect the nucleic acids from nuclease degradation without entirely inhibiting their catalytic activity. Resistance to nuclease degradation can increase the half-life of these nucleic acids inside a cell and improve the overall effectiveness of nucleic acid catalysts. These modifications may also be used to facilitate efficient uptake of nucleic acid catalysts by cells, transport and localization of these nucleic acids within a cell, and help achieve an overall improvement in the efficacy of nucleic acid catalysts in vitro and in vivo.
The term xe2x80x9cchemical substitutionxe2x80x9d as used herein refers to any base, sugar and/or phosphate modification that will protect the nucleic acids from degradation by nucleases without inhibiting their catalytic activity entirely.
In a preferred embodiment, the invention features a nucleic acid catalyst comprising at least one L-nucleotide, wherein the L-nucleotide has the formula I: 
wherein, X is a nucleic acid base, which may be modified or unmodified, or H; Y is a phosphorus-containing group; R1 is H, OH or other 2xe2x80x2-modifications; R2 is a blocking group or a phosphorus-containing group.
A xe2x80x9cblocking groupxe2x80x9d is a group which is able to be removed after polynucleotide synthesis and/or which is compatible with solid phase polynucleotide synthesis.
A xe2x80x9cphosphorus containing groupxe2x80x9d can include phosphorus in forms such as dithioates, phosphoramidites and/or as part of an oligonucleotide.
In one preferred embodiment the invention features a nucleic acid catalyst made up entirely of L-nucleotides of Formula I and has no D-nucleotide residue (L-nucleic acid catalyst). More specifically, the L-nucleic acid catalyst is an RNA or a DNA or combinations of ribo- and deoxyribonucleotides. Alternately, or in addition, the L-nucleic acid catalyst is modified at the base, sugar, and/or phosphate backbone individually or in combinations without entirely inhibiting the catalytic activity.
In another preferred embodiment the invention features a nucleic acid catalyst comprising at least two L-nucleotide substitutions of formula I, wherein said substitution is same or different.
In yet another preferred embodiment, the invention features a nucleic acid catalyst with L-nucleotide substitution of Formula I, wherein said nucleic acid can cleave a separate nucleic acid molecule, preferably a single-stranded nucleic acid, more specifically RNA.
In a preferred embodiment the invention features a nucleic acid catalyst with L-nucleotide substitution of Formula I, wherein the catalyst is in a hammerhead or a hairpin ribozyme motif.
In another aspect, the invention features a nucleic acid catalyst with L-nucleotide substitution of Formula I, wherein said nucleic acid ligates separate nucleic acid molecules.
The invention also features a nucleic acid molecule catalyst with L-nucleotide substitution of Formula I, wherein said nucleic acid molecule cleaves or forms amide or peptide linkages.
The term xe2x80x9cnucleotidexe2x80x9d is used as recognized in the art to include natural bases, and modified bases well known in the art. Such bases are generally located at the 1xe2x80x2 position of a sugar moiety. Nucleotide generally comprise a base, sugar and a phosphate group. The nucleotides can be unmodified or modified at the sugar, phosphate and/or base moeity, (see for example, Usman and McSwiggen, supra; Eckstein et al., International PCT Publication No. WO 92/07065; Usman et al., International PCT Publication No. WO 93/15187; all hereby incorporated by reference herein). There are several examples of modified nucleic acid bases known in the art and has recently been summarized by Limbach et al., 1994, Nucleic Acids Res. 22, 2183. Some of the non-limiting examples of base modifications that can be introduced into enzymatic nucleic acids without significantly effecting their catalytic activity include, inosine, purine, pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2,4,6-trimethoxy benzene, 3-methyluracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidines (e.g., 5-methylcytidine), 5-alkyluridines (e.g., ribothymidine), 5-halouridine (e.g., 5-bromouridine) or 6-azapyrimidines or 6-alkylpyrimidines (e.g. 6-methyluridine) and others (Burgin et al., 1996, Biochemistry, 35, 14090). By xe2x80x9cmodified basesxe2x80x9d in this aspect is meant nucleotide bases other than adenine, guanine, cytosine and uracil at 1xe2x80x2 position or their equivalents; such bases may be used within the catalytic core of the enzyme and/or in the substrate-binding regions.
There are several examples in the art describing sugar modifications that can be introduced into enzymatic nucleic acid molecules without significantly effecting catalysis and significantly enhancing their nuclease stability and efficacy. Sugar modification of enzymatic nucleic acid molecules have been extensively described in the art (see Eckstein et al., International Publication PCT No. WO 92/07065; Perrault et al. Nature 1990, 344, 565-568; Pieken et al. Science 1991, 253, 314-317; Usman and Cedergren, Trends in Biochem. Sci. 1992, 17, 334-339; Usman et al. International Publication PCT No. WO 93/15187; Sproat, U.S. Pat. No. 5,334,711 and Beigelman et al., 1995 J. Biol. Chem. 270, 25702). Such publications describe the location of incorporation of modifications and the like, and are incorporated by reference herein. In view of such teachings, similar modifications can be used as described herein.
In a preferred embodiment the invention features a nucleic acid catalyst with non-nucleotide substitution. The non-nucleotide substituion are in addition to the L-nucleotide substitution and/or the non-nucleotide substitution is in the opposite enantiomeric form as the standard non-nucleotide residue. The term xe2x80x9cnon-nucleotidexe2x80x9d as used herein include either abasic nucleotide, polyether, polyamine, polyamide, peptide, carbohydrate, lipid or polyhydrocarbon compounds. Specific examples include those described by Seela and Kaiser, Nucleic Acids Res. 1990, 18:6353 and Nucleic Acids Res. 1987, 15:3113; Cload and Schepartz, J. Am. Chem. Soc. 1991, 113:6324; Richardson and Schepartz, J. Am. Chem. Soc. 1991, 113:5109; Ma et al., Nucleic Acids Res. 1993, 21:2585 and Biochemistry 1993, 32:1751; Durand et al., Nucleic Acids Res. 1990, 18:6353; McCurdy et al., Nucleosides and Nucleotides 1991, 10:287; Jschke et al., Tetrahedron Lett. 1993, 34:301; Ono et al., Biochemistry 1991, 30:9914; Arnold et al., International Publication No. WO 89/02439; Usman et al., International Publication No. WO 95/06731; Dudycz et al., International Publication No. WO 95/11910 and Ferentz and Verdine, J. Am. Chem. Soc. 1991, 113:4000, all hereby incorporated by reference herein. Thus, in a preferred embodiment, the invention features an enzymatic nucleic acid molecule having one or more non-nucleotide moieties, and having enzymatic activity to cleave an RNA or DNA molecule. By the term xe2x80x9cnon-nucleotidexe2x80x9d is meant any group or compound which can be incorporated into a nucleic acid chain in the place of one or more nucleotide units, including either sugar and/or phosphate substitutions, and allows the remaining bases to exhibit their enzymatic activity. The group or compound is abasic in that it does not contain a commonly recognized nucleotide base, such as adenosine, guanine, cytosine, uracil or thymine. The term xe2x80x9cabasicxe2x80x9d or xe2x80x9cabasic nucleotidexe2x80x9d as used herein encompasses sugar moieties lacking a base or having other chemical groups in place of base at the 1xe2x80x2 position.
In preferred embodiments, the enzymatic nucleic acid includes one or more stretches of RNA, which provide the enzymatic activity of the molecule, linked to the non-nucleotide moiety. The necessary RNA components are known in the art, see, e.g., Usman, supra. By RNA is meant a molecule comprising at least one ribonucleotide residue.
As the term is used in this application, non-nucleotide-containing enzymatic nucleic acid means a nucleic acid molecule that contains at least one non-nucleotide component which replaces a portion of a ribozyme, e.g., but not limited to, a double-stranded stem, a single-stranded xe2x80x9ccatalytic corexe2x80x9d sequence, a single-stranded loop or a single-stranded recognition sequence. These molecules are able to cleave (preferably, repeatedly cleave) separate RNA or DNA molecules in a nucleotide base sequence specific manner. Such molecules can also act to cleave intramolecularly if that is desired. Such enzymatic molecules can be targeted to virtually any RNA transcript.
By xe2x80x9cL-nucleotidexe2x80x9d is meant a nucleotide having the opposite rotatory dispersion spectra to their naturally occurring D-enantiomers (Rosanoff, supra). Enantiomers as used herein is meant to indicate the mirror images of each other, as defined by Jacques et al., 1991, Enantiomers, Racemates, and Resolutions, pp 3, Krieger Publishing Co., Florida, USA.
Jacques et al., 1991, Enantiomers, Racemates, and Resolutions, pp 3, Krieger Publishing Co., Florida, USA, define chirality, racemates and enantiomers. They state on pages 3-4 thatxe2x80x94
xe2x80x9cChirality is a concept well known to organic chemists and, indeed, to all chemists concerned in any way with structure. It has numerous implications ranging from those affecting physical properties of matter to those related to biological mechanisms. These implications extend far beyond the borders of xe2x80x9cpurexe2x80x9d chemistry . . . . xe2x80x9d
The geometric property that is responsible for the nonidentity of an object with its mirror image is called chirality. A chiral object may exist in two enantiomorphic forms which are mirror images of one another. Such forms lack inverse symmetry elements, that is, a center, a plane, and an improper axis of symmetry. Objects that possess one or more of these inverse symmetry elements are superposable on their mirror images; they are achiral. All objects necessarily belong to one of these categories; a hand, a spiral staircase, and a snail shell are all chiral, while a cube and a sphere are achiral.
. . . All of the foregoing definitions remain valid at the molecular level; there are achiral as well as chiral molecules. The latter exist in two enantiomeric forms (the adjective enantiomorphic is more generally applied to macroscopic objects). The term enantiomer is used to designate either a single molecule, a homochiral collection of molecules, or even a heterochiral collection that contains an excess of one enantiomer and whose composition is defined by its enantiomeric purity p, or the enantiomeric excess e.e. which is equivalent to p.
The oldest known manifestation of molecular chirality is the optical activity, or rotatory power, the property that is exhibited by the rotation of the plane of polarization of light. The two enantiomers of a given compound have rotatory powers of equal absolute value but of opposite sign, or sense. One is positive, or dextrorotatory, while the other is negative, or levorotatory. The absolute designations of sign are arbitrary inasmuch as they are wavelength, temperature, and solvent dependent, but the relative designations are always valid. That is, a given enantiomer may be (+) at one wavelength and (xe2x88x92) at another. The other enantiomer will always have the opposite sign at the corresponding wavelength. While we shall use as often as possible the (+) and (xe2x88x92) symbols to designate a pair of enantiomers, we shall occasionally employ the letters d and I or D and L for convenience.
. . . The absolute configuration of a chiral substance is known when an enantiomeric structure can be assigned to an optically active sample of a given sign . . . . Recall that absolute configurations are designated by means of an alphabetic symbolism (R, S for rectus and sinister) whose application is determined by the rules of Cahn, Ingold, and Prelog. However, the D and L descriptors of Rosanoff are still used for carbohydrates. Care should be exercised so as not to confuse these with the sign of the optical activity.xe2x80x9d (emphasis added)
Tazawa et al., 1970, Biochemistry, 3499, described the synthesis of dinucleotides with L-adenosine residues. They also reported that L-adenosine dimers are xe2x80x9ccompletelyxe2x80x9d or xe2x80x9cextremelyxe2x80x9d resistant to cleavage by spleen and snake venom phosphodiesterase enzymes.
Ashley, 1992, J. Am. Chem. Soc., 114, 9731, RNA molecules composed entirely of L-ribonucleotides {(L)-RNA} can interact stably with a complementary (D)-RNA and poorly with a complementary (D)-DNA. He also mentions in the paper that (L)-RNA is xe2x80x9cresistant to both purified ribonuclease A and total cell extracts of L-cells.xe2x80x9d
Klubmann et al., 1996, Nature Biotech., 14, 1112; Nolte et al., 1996, Nature Biotech., 14, 1116, describe a method of selecting L-oligonucleotide aptamers capable of binding D-adenosine and L-arginine ligands.
Schumacher and Kim, International PCT Publication No. WO 96/34879, describe a method of identifying xe2x80x9cmacromolecules (peptides, oligonucleotides, sugar and macromolecular complexes, such as RNA-protein complexes, protein-lipid complexes), which are not of the natural handedness (not of the chirality as they occur in nature or as wildtype molecule) and which are ligands for other chiral macromolecules.xe2x80x9d The publications cited above and elswhere in the application, describe general methods and strategies to determine the location of incorporation of sugar, base and/or phosphate modifications including the L-nucleotide substitution and the like into ribozymes wihout inhibiting catalysis, and are incorporated by reference herein. In view of such teachings, similar modifications can be used as described herein to modify the nucleic acid catalysts of the instant invention.
By the phrase xe2x80x9cnucleic acid catalystxe2x80x9d is meant a nucleic acid molecule capable of catalyzing a variety of reactions including the ability to repeatedly cleave other separate nucleic acid molecules (endonuclease activity) in a nucleotide base sequence-specific manner. Such a molecule with endonuclease activity may have complementarity in a substrate binding region to a specified gene target, and also has an enzymatic activity that specifically cleaves RNA or DNA in that target. That is, the nucleic acid molecule with endonuclease activity is able to intramolecularly or intermolecularly cleave RNA or DNA and thereby inactivate a target RNA or DNA molecule. This complementarity functions to allow sufficient hybridization of the enzymatic RNA molecule to the target RNA or DNA to allow the cleavage to occur. 100% complementarity is preferred, but complementarity as low as 50-75% may also be useful in this invention. The nucleic acids may be modified at the base, sugar, and/or phosphate groups. The term enzymatic nucleic acid is used interchangeably with phrases such as ribozymes, catalytic RNA, enzymatic RNA, catalytic DNA, nucleozyme, DNAzyme, RNA enzyme, endoribonuclease, endonuclease, minizyme, leadzyme, oligozyme or DNA enzyme. All of these terminologies describe nucleic acid molecules with enzymatic activity.
By xe2x80x9cnucleic acid moleculexe2x80x9d as used herein is meant a molecule comprising nucleotides. The nucleic acid can be composed of modified or unmodified nucleotides or non-nucleotides or various mixtures and combinations thereof.
By xe2x80x9ccomplementarityxe2x80x9d is meant a nucleic acid that can form hydrogen bond(s) with other RNA sequence by either traditional Watson-Crick or other non-traditional types (for example, Hoogsteen type) of base-paired interactions.
In preferred embodiments of this invention, the enzymatic nucleic acid molecule is formed in a hammerhead or hairpin motif, but may also be formed in the motif of a hepatitis delta virus (HDV), group I intron, RNaseP RNA (in association with an RNA guide sequence) or Neurospora VS RNA. Examples of such hammerhead motifs are described by Rossi et al., 1992, Aids Research and Human Retroviruses 8, 183, of hairpin motifs by Hampel et al., EP0360257, Hampel and Tritz, 1989 Biochemistry 28, 4929, and Hampel et al., 1990 Nucleic Acids Res. 18, 299, and an example of the hepatitis delta virus motif is described by Perrotta and Been, 1992 Biochemistry 31, 16; of the RNaseP motif by Guerrier-Takada et al., 1983 Cell 35, 849 and Forster and Altman, 1990 Science 249, 783, Neurospora VS RNA ribozyme motif is described by Collins (Saville and Collins, 1990 Cell 61, 685-696; Saville and Collins, 1991 Proc. Natl. Acad. Sci. USA 88, 8826-8830; Guo and Collins, 1995 EMBO J. 14, 368) and of the Group I intron by Cech et al., U.S. Pat. No. 4,987,071. These specific motifs are not limiting in the invention and those skilled in the art will recognize that all that is important in an enzymatic nucleic acid molecule with endonuclease activity of this invention is that it has a specific substrate binding site which is complementary to one or more of the target gene RNA regions, and that it have nucleotide sequences within or surrounding that substrate binding site which impart an RNA cleaving activity to the molecule.
The invention provides a method for producing a class of enzymatic cleaving agents which exhibit a high degree of specificity for the target, such as RNA of a desired target. The enzymatic nucleic acid molecule is preferably targeted to a highly conserved sequence region of a target such that specific treatment of a disease or condition can be provided with a single enzymatic nucleic acid. Such enzymatic nucleic acid molecules can be delivered exogenously to specific cells as required. In the preferred hammerhead motif the small size (less than 60 nucleotides, preferably between 30-40 nucleotides in length) of the molecule allows the cost of treatment to be reduced compared to other ribozyme motifs.
Therapeutic ribozymes must remain stable within cells until translation of the target mRNA has been inhibited long enough to reduce the levels of the undesirable protein. This period of time varies between hours to days depending upon the disease state. Clearly, ribozymes must be resistant to nucleases in order to function as effective intracellular therapeutic agents. Improvements in the chemical synthesis of RNA (Wincott et al., 1995 Nucleic Acids Res. 23, 2677; incorporated by reference herein) have expanded the ability to modify ribozymes to enhance their nuclease stability. The majority of this work has been performed using hammerhead ribozymes (reviewed in Usman and McSwiggen, 1995 supra) and can be readily extended to other catalytic nucleic acid motifs.
By xe2x80x9cenzymatic portionxe2x80x9d is meant that part of the ribozyme essential for cleavage of an RNA substrate.
By xe2x80x9csubstrate binding armxe2x80x9d is meant that portion of a ribozyme which is complementary to (i.e., able to base-pair with) a portion of its substrate. Generally, such complementarity is 100%, but can be less if desired. For example, as few as 10 bases out of 14 may be base-paired. Such arms are shown generally in FIG. 1 as discussed below. That is, these arms contain sequences within a ribozyme which are intended to bring ribozyme and target RNA together through complementary base-pairing interactions; e.g., ribozyme sequences within stems I and III of a standard hammerhead ribozyme make up the substrate-binding domain (see FIG. 1).
In a preferred embodiment, the enzymatic nucleic acid molecules are added directly, or can be complexed with cationic lipids, packaged within liposomes, or otherwise delivered to smooth muscle cells. The RNA or RNA complexes can be locally administered to relevant tissues through the use of a catheter, infusion pump or stent, with or without their incorporation in biopolymers. Using the methods described herein, other enzymatic nucleic acid molecules that cleave target nucleic acid may be derived and used as described above. Specific examples of nucleic acid catalysts of the instant invention are provided below in the Tables and figures.
Sullivan, et al., supra, describes the general methods for delivery of enzymatic RNA molecules. Ribozymes may be administered to cells by a variety of methods known to those familiar to the art, including, but not restricted to, encapsulation in liposomes, by iontophoresis, or by incorporation into other vehicles, such as hydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres. For some indications, ribozymes may be directly delivered ex vivo to cells or tissues with or without the aforementioned vehicles. Alternatively, the RNA/vehicle combination is locally delivered by direct injection or by use of a catheter, infusion pump or stent. Other routes of delivery include, but are not limited to, intravascular, intramuscular, subcutaneous or joint injection, aerosol inhalation, oral (tablet or pill form), topical, systemic, ocular, intraperitoneal and/or intrathecal delivery. More detailed descriptions of ribozyme delivery and administration are provided in Sullivan et al., supra and Draper et al., supra which have been incorporated by reference herein.
By xe2x80x9cconsists essentially ofxe2x80x9d is meant that the active ribozyme contains an enzymatic center or core equivalent to those in the examples, and binding arms able to bind target nucleic acid molecules such that cleavage at the target site occurs. Other sequences may be present which do not interfere with such cleavage.
Thus, in one aspect, the invention features ribozymes that inhibit gene expression and/or cell proliferation. These chemically or enzymatically synthesized nucleic acid molecules contain substrate binding domains that bind to accessible regions of specific target nucleic acid molecules. The nucleic acid molecules also contain domains that catalyze the cleavage of target. Upon binding, the enzymatic nucleic acid molecules cleave the target molecules, preventing for example, translation and protein accumulation. In the absence of the expression of the target gene, cell proliferation, for example, is inhibited.
By xe2x80x9cpatientxe2x80x9d is meant an organism which is a donor or recipient of explanted cells or the cells themselves. xe2x80x9cPatientxe2x80x9d also refers to an organism to which enzymatic nucleic acid molecules can be administered. Preferably, a patient is a mammal or mammalian cells. More preferably, a patient is a human or human cells.
In a preferred embodiment, the invention features a method of synthesis of enzymatic nucleic acid molecules of instant invention which follows the procedure for normal chemical synthesis of RNA as described in Usman et al., 1987 J. Am. Chem. Soc., 109, 7845; Scaringe et al., 1990 Nucleic Acids Res., 18, 5433; and Wincott et al., 1995 Nucleic Acids Res. 23, 2677-2684 and makes use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5xe2x80x2-end, and phosphoramidites at the 3xe2x80x2-end. Small scale synthesis were conducted on a 394 Applied Biosystems, Inc. synthesizer using a modified 2.5 xcexcmol scale protocol with a 5 min coupling step for alkylsilyl protected nucleotides and 2.5 min coupling step for 2xe2x80x2-O-methylated nucleotides. Table II outlines the amounts, and the contact times, of the reagents used in the synthesis cycle. A 6.5-fold excess (163 xcexcL of 0.1 M=16.3 xcexcmol) of phosphoramidite and a 24-fold excess of S-ethyl tetrazole (238 xcexcL of 0.25 M=59.5 xcexcmol) relative to polymer-bound 5xe2x80x2-hydroxyl is used in each coupling cycle. Average coupling yields on the 394 Applied Biosystems, Inc. synthesizer, determined by colorimetric quantitation of the trityl fractions, is 97.5-99%. Other oligonucleotide synthesis reagents for the 394 Applied Biosystems, Inc. synthesizer: detritylation solution was 2% TCA in methylene chloride (ABI); capping was performed with 16% N-methyl imidazole in THF (ABI) and 10% acetic anhydride/10% 2,6-lutidine in THF (ABI); oxidation solution was 16.9 mM 12, 49 mM pyridine, 9% water in THF (Millipore). B and J Synthesis Grade acetonitrile is used directly from the reagent bottle. S-Ethyl tetrazole solution (0.25 M in acetonitrile) is made up from the solid obtained from American International Chemical, Inc.
In a preferred embodiment, deprotection of the chemically synthesized nucleic acid catalysts of the invention is performed as follows. The polymer-bound oligoribonucleotide, trityl-off, is transferred from the synthesis column to a 4 mL glass screw top vial and suspended in a solution of methylamine (MA) at 65xc2x0 C. for 10 min. After cooling to xe2x88x9220xc2x0 C., the supernatant is removed from the polymer support. The support is washed three times with 1.0 mL of EtOH:MeCN:H2O/3:1:1, vortexed and the supernatant is then added to the first supernatant. The combined supernatants, containing the oligoribonucleotide, are dried to a white powder.
The base-deprotected oligoribonucleotide is resuspended in anhydrous TEA.HF/NMP solution (250 xcexcL of a solution of 1.5 mL N-methylpyrrolidinone, 750 xcexcL TEA and 1.0 mL TEA.3HF to provide a 1.4M HF concentration) and heated to 65xc2x0 C. for 1.5 h. The resulting, fully deprotected, oligomer is quenched with 50 mM TEAB (9 mL) prior to anion exchange desalting.
For anion exchange desalting of the deprotected oligomer, the TEAB solution is loaded on to a Qiagen 500(copyright) anion exchange cartridge (Qiagen Inc.) that is prewashed with 50 mM TEAB (10 mL). After washing the loaded cartridge with 50 mM TEAB (10 mL), the RNA is eluted with 2 M TEAB (10 mL) and dried down to a white powder. The average stepwise coupling yields are generally  greater than 98% (Wincott et al., 1995 Nucleic Acids Res. 23, 2677-2684).
In a preferred embodiment, the ribozymes of the instant invention are also synthesized from DNA templates using bacteriophage T7 RNA polymerase (Milligan and Uhlenbeck, 1989, Methods Enzymol. 180, 51).
In another embodiment, the ribozymes are purified by gel electrophoresis using general methods or are purified by high pressure liquid chromatography (HPLC; See Wincott et al., supra) the totality of which is hereby incorporated herein by reference) and are resuspended in water.
In a preferred embodiment, in addition to L-nucleotide substitution, the nucleic acid catalysts of the invention are modified to enhance stability and/or enhance catalytic activity by modification with nuclease resistant groups, for example, 2xe2x80x2-amino, 2xe2x80x2-C-allyl, 2xe2x80x2-flouro, 2xe2x80x2-O-methyl, 2xe2x80x2-H, nucleotide base modifications (for a review see Usman and Cedergren, 1992 TIBS 17, 34; Usman et al., 1994 Nucleic Acids Symp. Ser. 31, 163; Burgin et al., 1996 Biochemistry 35, 14090).
In a preferred embdiment the molecules of the invention are purified by gel electrophoresis using general methods or are purified by high pressure liquid chromatography (HPLC; See Wincott et al., supra) the totality of which is hereby incorporated herein by reference) and are resuspended in water.
In another preferred embodiment, catalytic activity of the molecules described in the instant invention can be optimized as described by Draper et al., supra. The details will not be repeated here, but include altering the length of the ribozyme binding arms, or chemically synthesizing ribozymes with modifications (base, sugar and/or phosphate) that prevent their degradation by serum ribonucleases and/or enhance their enzymatic activity (see e.g., Eckstein et al., International Publication No. WO 92/07065; Perrault et al., 1990 Nature 344, 565; Pieken et al., 1991 Science 253, 314; Usman and Cedergren, 1992 Trends in Biochem. Sci. 17, 334; Usman et al., International Publication No. WO 93/15187; and Rossi et al., International Publication No. WO 91/03162; Sproat, U.S. Pat. No. 5,334,711; and Burgin et al., supra; all of these describe various chemical modifications that can be made to the base, phosphate and/or sugar moieties of enzymatic RNA molecules). Modifications which enhance their efficacy in cells, and removal of bases from stem loop structures to shorten RNA synthesis times and reduce chemical requirements are desired. (All these publications are hereby incorporated by reference herein.).
By xe2x80x9cenhanced enzymatic activityxe2x80x9d is meant to include activity measured in cells and/or in vivo where the activity is a reflection of both catalytic activity and ribozyme stability. In this invention, the product of these properties is increased or not significantly (less that 10 fold) decreased in vivo compared to an all RNA ribozyme.
In yet another preferred embodiment, nucleic acid catalysts having chemical modifications which maintain or enhance enzymatic activity is provided. Such nucleic acid is also generally more resistant to nucleases than unmodified nucleic acid. Thus, in a cell and/or in vivo the activity may not be significantly lowered. As exemplified herein such ribozymes are useful in a cell and/or in vivo even if activity over all is reduced 10 fold (Burgin et al., 1996, Biochemistry, 35, 14090). Such ribozymes herein are said to xe2x80x9cmaintainxe2x80x9d the enzymatic activity on all RNA ribozyme.
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.
The drawings will first briefly be described.